Separations based analyses are a prominent part of biological research, allowing one to characterize different biological samples, reaction products and the like. Examples of some of the more prevalent separations based analyses include electrophoretic separations of macromolecular species, e.g., proteins and nucleic acids. Capillary electrophoresis has been established as a highly effective method for separating macromolecular species in order that they might be further characterized. Protein and nucleic acid molecules are two major examples of molecular species that are routinely fractionated and characterized using capillary electrophoretic systems. These systems have generally proven effective as a result of the high surface to volume ratio of the thin capillaries. This high surface to volume ratio allows for much greater heat dissipation, which in turn, allows application of greater electrical fields to the capillary thereby resulting in a much more rapid separation of macromolecules introduced into the system.
Microfluidic devices have been applied in separations based analyses, and have yielded substantial advantages in speed and accuracy. Examples of novel microfluidic devices and methods for use in the separation of molecular, and particularly macromolecular species by electrophoretic means are described in U.S. Pat. Nos. 5,958,694 and 6,032,710, for example, the entire contents of which are incorporated by reference herein. In such devices, the sample containing the macromolecular species for which separation is desired, is placed in one end of a separation channel located in the microfluidic substrate and a voltage gradient is applied along the length of the channel. As the sample components are electrophoretically transported along the length of the channel and through the separation (sieving) matrix disposed therein, those components are resolved. The separated components are then detected at a detection point along the length of the channel, typically near the terminus of the separation channel downstream from the point at which the sample was introduced. Following detection, the separated components are typically directed to a collection reservoir/well in the device (or to an external device such as a multiwell plate via a capillary pipettor, for example) for subsequent extraction or disposal.
In many situations, it is desirable to extract selected fragments of interest following the separation of the fragments into bands in the separation matrix, such as DNA fragments for further processing or analysis, e.g., restriction enzyme modification, T4 ligation, PCR amplification, mass spectroscopy, or polynucleotide kinase reactions. The typical process used by laboratory researchers for extracting and isolating selected DNA fragments of interest (and other desired nucleic acid and protein fragments) from a separation matrix (such as agarose gels) involves manually transferring the DNA fragments to a suitable transfer medium, where the separated fragments are stained and illuminated by shining ultraviolet (UV) light on the fragments to visualize the separated bands. A razor blade is then used to manually cut above and below each fragment of interest. The recovered DNA can then be used for further processing or analysis. Such extraction process, however, is time consuming, laborious and potentially damaging to the DNA (e.g., nicking of the DNA can occur if the DNA is exposed to ultraviolet light too long while the fragments of interest are being illuminated for excision).
Thus, in performing separations based analyses in microfluidic devices, for example, it would be desirable to not only be able to rapidly collect data regarding the relative size and/or molecular weights (based on comparisons to standards, for example) of the separated components, but it would also be desirable to be able to isolate or extract one or more of the separated components in the device itself for further analysis or processing in the device, since the microscale dimensions of the device offer advantages in terms of automation, speed, reduced consumption of expensive reagents (typically on the order of nanoliters), and more efficient use of manpower as well as increased throughput. The recovered or isolated fragments could then be used for a variety of different processes in the device including, for example, ligation reactions for cloning small to medium-sized strands of DNA into bacterial plasmids, bacteriophages, and small animal viruses to allow the production of pure DNA in sufficient quantities to allow its chemical analysis, reactions to dissolve a separated protein or nucleic acid component in a suitable matrix for further analysis by a mass spectrometer using, for example, Matrix-Assisted Laser Desorption Ionization (MALDI), binding reactions to bind a labeling agent to one or more separated protein or nucleic acid components for further analysis, or other similar post-detection processes. In addition, in the case of polymerase chain reaction (PCR) samples, it is important to be able to separate smaller dimer and primer molecules from the main nucleic acid fragments in the sample and then isolate and collect the main nucleic acid fragments for further analysis or processing, while directing the smaller primer and dimer components to a waste reservoir/cell for removal and subsequent disposal.
Thus, it would be advantageous to provide improved microfluidic devices, systems and methods for use in separating sample materials into different sample components or fragments and then isolating one or more of the sample components for further processing or analysis in the device. Such devices preferably should employ configurations that optionally allow a sample material to be electrophoretically separated into sample components in a separation matrix within a separation conduit in the device. The sample components may then be detected in a detection zone in the separation conduit, and then selected fragments or components of interest shunted to a component collection conduit within the device downstream of the detection zone for further processing or analysis based on information (such as size-based information) received at the detection zone.